Neutral endopeptidase 24.11 (NEP) is a cell-surface peptidase expressed by prostatic epithelial cells which cleaves and inactivates neuropeptides implicated in the growth of androgen-independent prostate cancer (PC). We show that NEP expression and catalytic activity are lost in vitro in androgen-independent but not androgen-dependent PC cell lines. In vivo, NEP protein expression is commonly decreased in cancer cells of metastatic PC specimens from patients with androgen-independent but not androgen-dependent PC. Overexpression of NEP in androgen- independent PC cells or incubation with recombinant NEP inhibits PC cell growth. Furthermore, in androgen-dependent PC cells, expression of NEP is transcriptionally regulated by androgen and decreases with androgen- withdrawal. Consequently, PC cells which survive androgen-withdrawal can emerge with reduced NEP. These data suggest that decreased NEP expression, common in androgen-independent PCS, is facilitated by the elimination of androgens, and that NEP loss plays an important role in the development of androgen-independent PC by allowing PC cells to use mitogenic neuropeptides as an alternate source to androgen to stimulate cell proliferation. To thoroughly define the involvement of NEP on androgen-independent PC cells, our specific aims are (1) to explore the mechanism by which NEP inhibits cell growth; (2) to establish that the androgen response element (ARE) in the 3' end of the NEP gene is a functional ARE which enhances transcription of the NEP gene; and (3) to assess the antitumor effects of NEP in an animal model of prostate cancer by establishing that recombinant NEP can inhibit the tumorigenicity of androgen-independent PC cells in an orthotopic model of PC, and to establish that overexpression of NEP in androgen- independent PC cells inhibits the tumorigenicity of androgen-independent PC cells. These studies leading to a better understanding of the involvement of NEP in the development and progression of androgen- independent PC may ultimately provide support for novel approaches for the treatment of advanced PC.